Team:Paris Saclay/Notebook/August/9

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==='''A - Aerobic/Anaerobic regulation system'''===
==='''A - Aerobic/Anaerobic regulation system'''===
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((((((((((((((((((((===='''1 - Obtaining Δ ''fnr E. coli'' strain by transduction to test our biobricks'''====
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===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
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Abdou, Anais, Damir, Nadia, XiaoJing
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===='''1 - Tranduction of Km in MG1655Z1 ====
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We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain  was too old ( 2001) 
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Abdou, Anaïs, Damir, Nadia, XioaJing
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Protocol : [[Team:Paris_Saclay/Protocols/transduction|transduction]]
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We observed lysis areas. We will continue the transduction protocol.
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* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.  
* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.  
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
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*We used a wild type strain to keep a stock and a Δ ''fnr E. coli'' strain to obtain phages that might have encapsidated a Δfnr::Km fragment.
 
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10µl,50µl and 100µl petri dishes are clear so phages are multiplied.
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Protocol : [[Team:Paris_Saclay/Protocols/transduction|Transduction]]
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We let the antibiotic over night to select the right strain. ))))))))))))))))))))))
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Our Mutant bacteria was called BW : Δfnr::Km.
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Our wild type bacteria was called MG1655Z1.
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We used kanamycine antibiotic.
===='''Objective : obtaining Bba_K1155007'''====
===='''Objective : obtaining Bba_K1155007'''====
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the extraction was good. We will sequence our plasmid.  
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The extraction was good. We will sequence our plasmid.  
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Revision as of 14:49, 28 September 2013

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Contents

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Tranduction of Km in MG1655Z1

Abdou, Anaïs, Damir, Nadia, XioaJing

We observed lysis areas. We will continue the transduction protocol.

Ps908transduction.jpg

Picture: lysed cells comparison.

  • 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
  • 50µl phage: the petri dish is clear, bacteria are lysed by phages.

Protocol : Transduction

Our Mutant bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1. We used kanamycine antibiotic.

Objective : obtaining Bba_K1155007

1 - Extraction of Bba_K115007 from DH5α

Abdou

Protocol : Hight copy plamid extraction

We used colonies number 10, 14 and 15.

Nanodrop

  • Bba_K1155007 in clone 10 : 38ng/µl
  • Bba_K1155007 in clone 14 : 48.5ng/µl
  • Bba_K1155007 in clone 15 : 52 ng/µl

The extraction was good. We will sequence our plasmid.

A - Aerobic/Anaerobic regulation system / B - PCB sensing system

Objective : Obtaining FNR and BphR2 proteins

1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification

IMAGE
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL of BphR2 Part I+1µl of 6X loading dye
  • Well 3 : 5µL of BphR2 Part II+1µl of 6X loading dye
  • Well 4 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye
  • Well 5 : 5µL of FNR Part I+1µl of 6X loading dye
  • Well 6 : 5µL of FRN Part II+1µl of 6X loading dye
  • Well 7: 5µL of RBS-FNR Part I+1µl of 6X loading dye
  • Gel : 0.8%

Expected size :

  • BphR2 Part I :
  • BphR2 Part II :
  • RBS-BphR2 Part I :
  • FNR Part I :
  • FNR Part II :
  • RBS-FNR PartI :

We lost all our PCR fragments. We will do the PCR again.

2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I

Anaïs, Damir, Nadia, XiaoJing

Used quantities :

  • Bphr2 Part I :
    • Oligo 54F : 1µL
    • Oligo 55R : 1µL
    • Buffer Phusion : 10µL
    • DNA of Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • Bphr2 Part II :
    • Oligo 56F : 1µL
    • Oligo 57R : 1µL
    • Buffer Phusion : 10µL
    • DNA Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • RBS-Bphr2 Part I :
    • Oligo 58F : 1µL
    • Oligo 57R : 1µL
    • Buffer Phusion : 10µL
    • DNA Pseudomonas pseudoalcaligenes : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part I :
    • Oligo 59F : 1µL
    • Oligo 60R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • FNR Part II :
    • Oligo 61F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL
  • RBS-FNR Part I :
    • Oligo 63F : 1µL
    • Oligo 62R : 1µL
    • Buffer Phusion : 10µL
    • DNA Escherichia coli : 1µL
    • dNTP : 1µL
    • Phusion : 0.5µL
    • H2O : 35.5µL

PCR Program :

  • BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :

PsPCRBphR23007.jpg

  • FNR Part I, FNR Part II, RBS-FNR Part I :

PsPCRFNR3007.jpg


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